Anti-human urothelial carcinoma of supercritical carbon dioxide extract of cinnamomum subavenium, and the preparation process and uses

ABSTRACT

What is disclosed in the invention is a preparation method of a supercritical  Cinnamomum subavenium  extract, which is made from the material, the dried stem of  C. subavenium.  The extract is obtained by extracting  C. subavenium  which is pulverized as particles with supercritical carbon dioxide fluid. The  C. subavenium  extract or its active ingredient, subamolide A, can be used to inhibit the growth of human urothelial carcinoma cell lines. In addition, the  C. subavenium  extract (or subamolide A) is able to synergistically inhibit the growth of human urothelial carcinoma cell lines with cisplatin (CDDP) or gemcitabine (Gem). Therefore, the  C. subavenium  extract (or subamolide A) can be an anticancer drug alone, or forms a pharmaceutical composition with CDDP (or Gem) to treat with cancers in respect of urinary system.

CROSS-REFERENCE TO RELATED APPLICATION AND CLAIM OF PRIORITY

The application claims the benefit of Taiwan Patent Application No. 100141688, filed on Nov. 15, 2011, in the Taiwan Intellectual Property Office, the disclosures of which are incorporated herein in their entirety by reference.

FIELD OF THE INVENTION

The present invention relates to a Cinnamomum subavenium extract. In particular, the present invention relates to a supercritical carbon dioxide extract of C. subavenium, and the preparation process and the uses thereof. The C. subavenium supercritical extract is effective in anti-human urothelial carcinoma.

BACKGROUND OF THE INVENTION

Cinnamomum subavenium belongs to Cinnamomum genus, Lauraceae family, an endemic species grown in the forest at an altitude of 500 to 1000 meters in Taiwan, and also is nominated as Cinnamomum randaiense, obtuseleaf cinnamon bark, Cinnamomum osmophloeum, etc. It is also called as fragrant cinnamon because of its barks and leaves having fragrance. The volatile oil refined from barks of C. subavenium may be the essence material for cosmetics, and the leaf oil refined from leaves thereof may be the raw material for food and cigarettes, or pesticides. In traditional Chinese medicine, C. subavenium also is used to treat a various of diseases, such as stomachache, chest pain, abdominal pain, hernia, diarrhea, rheumatism, nausea, vomiting and so on.

Taiwan patent publication No. 200924788 discloses a pesticide combination, including C. osmophloeum essence oil and other essence oils for food industries and for bacteriostatic activity. That patent application only discloses the formulation made by essence oils containing C. osmophloeum essence oil, whereas it does not disclose the extraction method of C. osmophloeum essence oil and the components therein.

Taiwan patent publication No. 200914036 discloses a skin-used agent for prevent biting midges, including C. osmophloeum, betel nut and other major components. However, it does not disclose the preparation method of C. osmophloeum and the components contained therein.

Since there is not the components in C. subavenium and its preparation method disclosed in the prior art, it is impossible for one skilled in the art to use the components of C. subavenium in the medicines, cosmetics and other fields.

It is therefore attempted by the applicant to deal with the above situation encountered in the prior art.

SUMMARY OF THE INVENTION

For extracting the various components from C. subavenium, avoiding the use of organic solvents and reducing energy consumed in the extraction process, C. subavenium is extracted by using supercritical CO₂, by the inventors of the present invention, to obtain the plural compounds wherein subamolide A ([(3Z,4R,5R)-3-tetradecylidene-4-hydroxy-5-methoxy-5-methylbutanolide]) is the major component.C. subavenium supercritical CO₂ extract can be effectively in inhibiting the growth of urothelial carcinoma and treating cancers.

In addition, the combination of alkylating agent (e.g. cisplatin (cis-diamminedichloridoplatinum; “CDDP”)) (or nucleoside analog (e.g. gemcitabine (4-amino-1-(2-deoxy-2,2-difluoro-(3-D-erythro-pentofuranosyl)-pyrimidin-2(1H)-on; “Gem”)) with C. subavenium supercritical CO₂ extract or the combination of alkylating agent (or nucleoside analog) with subamolide A would synergistically inhibit the growth of urothelial carcinoma. Therefore, alkylating agent and C. subavenium supercritical CO₂ extract (or subamolide A) can be prepared as a pharmaceutical composition, and nucleoside analog and C. subavenium supercritical CO₂ extract (subamolide A) can be prepared as another one, and both pharmaceutical compositions are used for treating human urothelial carcinoma or cancers relevant to the urinary system.

The present invention provides a preparation method for C. subavenium extract, including steps of: (a) drying C. subavenium plant; (b) pulverizing the plant into plural particles; and (c) extracting the plural particles with supercritical CO₂ to obtain the C. subavenium extract including subamolide A.

Preferably, the step (a) further includes step of (al) drying the stem of C. subavenium plant. The step (c) is performed at a pressure of 150 to 350 bar, a temperature of 45° C. to 55° C., a flow rate of the supercritical CO₂ from 4 to 6 L/hr and a packing density of material between 250 g/L and 320 g/L. In some embodiments, the step (c) is further performed at 250 bar, 45° C., the flow rate of 4 L/hr and the packing density of material of 320 g/L.

The present invention further provides a pharmaceutical composition of C. subavenium extract for treating cancer cells and/or growth inhibition of the cancer cells, and the pharmaceutical composition includes: a first component having subamolide A; and a second component being selected from a group consisting of monoterpene, sesquiterpene, sesquiterpene derivative, saturated fatty acid, butanolide, phytosterol, triterpene, phytosterone and a combination thereof.

Preferably, (1) monoterpene includes but not limit to eugenol; (2) sesquiterpine includes but not limit to α-cubebene, α-bergamotene, trans-α-bergamotene, γ-elemene, β-acoradene, α-zingiberene, cis-α-bisabolene, β-bisabolene, β-curcumene, δ-amorphene and trans-α-bisabolene; (3) sequiterpene derivative includes but not limit to cedr-8-ene, α-curcumene, nerolidol, caryophyllene oxide, humulene-1,2-epoxide, cubenol, τ-cadinol, α-cadinol, epi-β-cadinol, epi-β-bisabolol, epi-α-bisabolol, α-bisabalol and 1,10-dihydro nootkatone; (4) saturated fatty acid includes but not limit to ethyl palmitate; (5) butanolide includes but not limit to isolinderanolide B, linderanolide B and secosubamolide; (6) phytosterol includes but not limit to β-sitosterol and γ-sitosterol; (7) triterpene includes but not limit to betulin; and (8) phytosterone includes but not limit to sitostenone.

The present invention further provides a pharmaceutical composition, including: subamolide A with a first effective amount; and an ingredient with a second effective amount and being an alkylating agent or a nucleoside analog.

Preferably, subamolide A is a component of a supercritical CO₂ extract of C. subavenium or is a component of a methanol (MeOH) extract thereof. Alkylating agent includes but not limit to cisplatin, carboplatin (cis-diammine(1,1-cyclobutanedicar-boxylato)platinum (II)) and oxaliplatin ([(1R,2R)-cyclohexane-1,2-diamine]-(ethanedioato-O′,O′)platinum (II)), and nucleoside analog includes but not limit to deoxyadenosine analog, deoxycytidine analog, deoxyguanosine analog, deoxythymidine analog, deoxyuridine analog, 6-thiohypoxanthine and fluorouracil. One of the deoxycytidine analog is Gem.

The above objectives and advantages of the present invention will become more readily apparent to those ordinarily skilled in the art after reviewing the following detailed descriptions and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the apparatus for preparing C. subavenium supercritical CO₂ extract in the present invention.

FIG. 2 is a diagram showing the cytotoxicity of C. subavenium supercritical CO₂ extract on human urothelial carcinoma cell line NTUB 1 and normal urothelial cell line SV-HUC-1.

FIG. 3 is a diagram showing the cellular viability of subamolide A on human urothelial carcinoma cell lines NTUB 1 and T24, human prostate cancer cell line PC3, and normal urothelial cell line SV-HUC-1.

FIG. 4( a) is a diagram showing the percentage of each cell cycle phase after the subamolide A treatment on NTUB 1 cells.

FIG. 4( b) is a diagram showing the intracellular reactive oxygen species (ROS) M1 ratio after the subamolide A treatment on NTUB1 and SV-HUC-1 cells.

FIG. 5( a) is an immunoblotting spectrum showing the cellular protein expressions after the subamolide A treatment on NTUB 1 cells.

FIGS. 5( b) and 5(c) respectively are the diagrams showing the fold of changes of (b) Bax/Bcl-2 ratio and (c) cytochrome c after the subamolide A treatment on NTUB 1 cells.

FIGS. 6( a) and 6(b) are the immunoblotting spectra showing the protein expressions of NTUB 1 cells post the subamolide A treatment.

FIGS. 7( a) and 7(b) respectively are the diagrams showing (a) the cellular viability and (b) the combination index (CI) of the combinational cytotoxic effect of subamolide A with CDDP on NTUB 1 cells for 24, 48 and 72 hours. CI is obtained from the median-effect analysis performed by the computer software Calcusyn™.

FIGS. 7( c) and 7(d) respectively are the diagrams showing (c) the cellular viability and (d) the CI of the combinational cytotoxic effect of subamolide A with Gem on NTUB 1 cells for 24, 48 and 72 hours. CI is obtained from the median-effect analysis performed by the computer software Calcusyn™.

FIGS. 8( a) and 8(b) respectively are the diagrams showing (a) the cellular viability and (b) the CI of the combinational cytotoxic effect of C. subavenium supercritical CO₂ extract (containing the major component, subamolide A) with CDDP (or Gem) on NTUB1 cells for 24 hours. CI is obtained from the median-effect analysis performed by the computer software Calcusyn™.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention will now be described more specifically with reference to the following Embodiments. It is to be noted that the following descriptions of preferred Embodiments of this invention are presented herein for purpose of illustration and description only; it is not intended to be exhaustive or to be limited to the precise form disclosed.

Embodiment

C. subavenium extract (or nominated as C. subavenium MeOH extract) or C. subavenium supercritical CO₂ extract in the present invention can be used in inhibiting the growth of urothelial carcinoma and treating cancers. Alternatively, the major component, subamolide A, in C. subavenium extract or C. subavenium supercritical CO₂ extract can be prepared as an anticancer pharmaceutical extract. Alternatively, an effective amount of subamolide A and an effective amount of other ingredient also can be prepared as an anticancer pharmaceutical extract.

C. subavenium extract or C. subavenium supercritical CO₂ extract is able to synergistically inhibit the growth of urothelial carcinoma with an alkylating agent (an anticancer drug). Alternatively, subamolide A in C. subavenium extract or C. subavenium supercritical CO₂ extract is able to synergistically inhibit the growth of urothelial carcinoma with the alkylating agent.

C. subavenium extract or C. subavenium supercritical CO₂ extract is able to synergistically inhibit the growth of urothelial carcinoma with a nucleoside analog (an anticancer drug). Alternatively, subamolide A in C. subavenium extract or C. subavenium supercritical CO₂ extract is able to synergistically inhibit the growth of urothelial carcinoma with the nucleoside analog.

The terms, e.g. “extract”, “compound”, “alkylating agent”, “analog”, “pharmaceutical composition”, “extract” and so on, herein have an effective amount upon usage, indicating an minimum effective amount for cytotoxicity, an effective amount within a scope or an maximum effective amount.

Alkylating agent includes but not limit to cisplatin, carboplatin and oxaliplatin, nucleoside analog includes but not limit to deoxyadenosine analog, deoxycytidine analog (e.g. Gem), deoxyguano sine analog, deoxythymidine analog, deoxyuridine analog, 6-thiohypoxanthine and fluorouracil, etc.

Experiments and Results:

I. Preparation and Analysis of C. Subavenium Supercritical CO₂ Extract

C. subavenium supercritical CO₂ extract of the present invention was made by extracting C. subavenium with supercritical CO₂ . C. subavenium's stems could be adopted as the material for extraction. First, the dried stems of C. subavenium (5 kg) were mechanically or physically pulverized as particles with average diameter of 1˜2 mm. Next, the experiment design was performed by using the orthogonal array of Taguchi methodology. The orthogonal array L9(3⁴) was selected for permutation and combination in the present invention, and 4 control factors were determined. Control factor A was extraction pressure, control factor B was extraction temperature, control factor C was flow rate of supercritical CO₂ fluid mass, and control factor D was packing density of material. The parameters for determining each control factor were described as follows. Extraction pressure was 150, 250 and 350 bar respectively, extraction temperature was 45, 50 and 55° C. respectively, flow rate of supercritical CO₂ fluid mass was 4, 5 and 6 L/hr respectively, and packing density of material was 250, 285 and 320 g/L respectively.

Next, please refer to apparatus diagram in FIG. 1, the pulverized C. subavenium particles were disposed in supercritical extraction vessel 5, and solid extraction was processed with supercritical CO₂ fluid. After CO₂ within the CO₂ reservoir 1 was pre-cooled at pre-cooler 2 and then was transmitted through high-pressure diaphragm pump 3 and heater 4, CO₂ entered into supercritical extraction vessel 5 by setting the aforementioned control factors (operation pressure of 150˜350 bar, temperature of 45° C.˜55° C., flow rate of 4 L/hr˜6 L/hr). C. subavenium supercritical CO₂ extract post the solid extraction and the supercritical CO₂ fluid were conducted into gas-liquid separator 6, and C. subavenium supercritical CO₂ extract was harvested at the bottom outlet of gas-liquid separator 6. The phase of supercritical CO₂ fluid was changed as gas to reduce the solubility of C. subavenium supercritical CO₂ extract, so that C. subavenium supercritical CO₂ extract was separated from supercritical CO₂ fluid and gas-liquid separation was achieved. The gaseous CO₂ was pressured and sent to CO₂ reservoir 1 for recycling.

C. subavenium supercritical CO₂ extract was analyzed to afford 34 components (Table 1) using gas chromatography-mass spectrometry (GC-MS) known by one skilled in the art. Components in Table 1 can be divided as monoterpene (e.g. eugenol), sesquiterpine (e.g. α-cubebene, α-bergamotene, trans-α-bergamotene, γ-elemene, β-acoradene, α-zingiberene, cis-α-bisabolene, β-bisabolene, β-curcumene, β-amorphene and trans-α-bisabolene), sequiterpene derivative (e.g. cedr-8-ene, α-curcumene, nerolidol, caryophyllene oxide, humulene-1,2-epoxide, cubenol, τ-cadinol, α-cadinol, epi-β-cadinol, epi-β-bisabolol, epi-α-bisabolol, α-bisabalol and 1,10-dihydro nootkatone), saturated fatty acid (e.g. ethyl palmitate), butanolide (e.g. isolinderanolide B, linderanolide B, secosubamolide and subamolide A), phytosterol (e.g. β-sitosterol and γ-sitosterol), triterpene (e.g. betulin) and phytosterone (e.g. sitostenone).

Since C. subavenium supercritical CO₂ extract are extracted from C. subavenium, the types of components, and the species and amounts of compounds are influenced by factors such as climate, temperature, moisture, rainwater, cultivation environment, soil, harvest and so on. Therefore, the components and ratios of C. subavenium supercritical CO₂ extracts are diverse with the batches. One skilled in the art can arbitrarily prepare the pharmaceutical extract including subamolide A and/or other ingredients in accordance with the types and ingredient of these components based on the present invention.

TABLE 1 Analysis of components in C. subavenium supercritical CO₂ extract using GC-MS Percent- Identifi- No. Components KI^(a) KI^(b) age (%)^(c) cation^(d) 1 Eugenol 1341 1341 1.29 MS, KI 2 α-Cubebene 1363 1360 0.48 MS, KI 3 Cedr-8-ene 1401 1405 0.28 MS, KI 4 α-Bergamotene 1394 1394 0.17 MS, KI 5 trans-α-Bergamotene 1423 1423 0.68 MS, KI 6 γ-Elemene 1438 1433 1.31 MS, KI 7 β-Acoradiene 1442 1440 0.36 MS, KI 8 α-Curcumene 1470 1470 6.27 MS, KI 9 α-Zingiberene 1495 1495 1.66 MS, KI 10 Cis-α-bisabolene 1494 1494 0.66 MS, KI 11 β-Bisabolene 1500 1500 4.66 MS, KI 12 β-Curcumene 1503 1503 0.56 MS, KI 13 δ-Amorphene 1499 1499 1.03 MS, KI 14 trans-α-Bisabolene 1523 1530 1.79 MS, KI 15 Nerolidol 1549 1550 0.36 MS, KI 16 Caryophyllene oxide 1551 1549 0.86 MS, KI 17 Humulene-1,2-epoxide 1581 1580 0.73 MS, KI 18 Cubenol 1603 1601 2.79 MS, KI 19 τ-Cadinol 1626 1625 2.00 MS, KI 20 α-Cadinol 1628 1628 0.84 MS, KI 21 epi-β-Cadinol 1641 1644 1.74 MS, KI 22 epi-β-Bisabolol 1669 1672 14.79 MS, KI 23 epi-α-Bisabolol 1688 1687 0.78 MS, KI 24 α-Bisabalol 1690 1691 0.57 MS, KI 25 1,10-Dihydro nootkatone 1756 1761 0.63 MS, KI 26 Ethyl palmitate 1982 1983 0.61 MS, KI 27 Isolinderanolide B 2291 — 10.29 MS 28 Linderanolide B 2326 — 1.22 MS 29 Secosubamolide 2410 — 3.42 MS 30 Subamolide A 2427 — 35.10 MS, ST 31 β-Sitosterol 3317 — 1.28 MS 32 γ-Sitosterol 3363 — 0.39 MS 33 Betulin 3380 — 0.13 MS 34 Sitostenone 3487 — 0.30 MS ^(a)Kovats index relative to n-alkanes (C₁₀-C₄₀) on a DB-5MS column. ^(b)Reference kovats index on a DB-5MS column. ^(c)Relative percentage calculated by integrated peak area in Thermo Xcalibur ™ data analysis program. ^(d)Identification based on comparison of the mass spectrum, co-injected with standard & Kovats index on a DB-5MS column in reference.

It could be known from Table 1 that subamolide A ([(3Z,4R,5R)-3-tetradecylidene-4-hydroxy-5-methoxy-5-methylbutanolide; formula I; 35.1% of percentage) was the major component in C. subavenium supercritical CO₂ extract.

The experimental design in the present invention was processed using the orthogonal array L9(3⁴) of Taguchi methodology to establish the best operational condition with extraction pressure of 150 bar, extraction temperature of 45° C., flow rate of supercritical CO₂ fluid mass of 4 L/hr and packing density of material of 250 g/L. It could be known from the signal to noise ratio (SN ratio) that the major factors to influence the yield of C. subavenium supercritical CO₂ extract sequentially were flow rate of supercritical CO₂ fluid mass, extraction temperature, packing density of material and extraction pressure, and the best predicted operational condition were extraction pressure of 250 bar, extraction temperature of 45° C., flow rate of supercritical CO₂ fluid mass of 4 L/hr and packing density of material of 320 g/L. Yields obtained from the above two operational conditions were 7.7% and 7.8% respectively, indicating that the operational conditions established from two experimental methods were similar, and confirming that the difference between the experimental result and the predicted SN ratio was less than 5. That is, the selected experimental condition was established, and the yield of supercritical extraction was 5.39% to be better than yield of organic solvent extraction of 1.54%.

Cytotoxicity assay was performed by using C. subavenium supercritical CO₂ extract obtained according to the preparation method mentioned above. Please refer to FIG. 2, it was found that C. subavenium supercritical CO₂ extract showed the best cytotoxicity (IC₅₀=0.67±0.02 μg/mL) to human urothelial carcinoma cell line NTUB 1 and the cytotoxicity (IC₅₀=14.1±1.15 μg/mL) to normal urothelial cell line SV-HUC-1. C. subavenium supercritical CO₂ extract selectively functioned on NTUB 1 carcinoma cells rather than the normal cells.

II. Preparation of C. Subavenium Extract

Additionally, subamolide A (purity >90%) was isolated from the stems of C. subavenium. Briefly, the air-dried stems were extracted with MeOH at room temperature. The MeOH extract, obtained by concentration under reduced pressure, was suspended in H₂O and then partitioned with chloroform (CHCl₃) to yield fractions soluble in CHCl₃ and H₂O. The CHCl₃ soluble fraction was chromatographed over silica gel using n-hexane-ethyl acetate (EtOAc)-MeOH mixtures as eluents and separated into five fractions. Fraction 2 was re-subjected to silica gel column chromatography and purified by preparative thin layer chromatography using n-hexane-EtOAc to yield subamolide A. Subamolide A was dissolved in dimethyl sulfoxide (DMSO) and stored at −20° C.

III. Statistical Analysis

Data of the following experimental results were expressed as means ±SD. Statistical analyses were performed using the Bonferroni t-test method after ANOVA for multigroup comparison and the Student's t-test method for two group comparison, with *P<0.05, **P<0.01 and ***P<0.001 were considered to be statistically significant.

IV. Cytotoxic Effect of Subamolide A on Various Cell Lines

Cellular cytotoxicity of tested compounds was performed by using a MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Briefly, the cells were plated with a density of 1×10³ cells/well in 96-well plates and incubated at 37° C. overnight before drug treatment. Cells were then cultured in the absence or presence of various concentrations (0.3, 1, 3, 6, 9, 12, 15 and 20 μM) of subamolide A at 37° C. for 72 hours. Subsequently, 50 μL of MTT (2 mg/mL in PBS) was added to each well and allowed to react for another 4 hours. Following centrifugation at 1000×g for 10 minutes, media were removed and 150 μL DMSO were added to each well. The proportions of surviving cells were determined by absorbance spectrometry at 540 nm using a microplate reader. The cell viability was expressed as the survival ratio to the un-treated control. The IC₅₀ values of each group were calculated by the median inhibitory analysis and presented as means±SD. The combinational effects of two compounds were analyzed by median-effect analysis. To evaluate the combined effects in growth inhibition, a corresponding combination index (CI) was adopted for the measurement. The combination index of <1, =1, or >1 denotes synergic, additive, or antagonistic effect, respectively.

Please refer to FIG. 3, which is the diagram showing the cellular cytotoxicity of subamolide A on human urothelial carcinoma cell line. Subamolide A showed a significant reduction of growth in urothelial carcinoma cells NTUB1 (IC₅₀=7.26±0.67 μM) and T24 (IC₅₀=7.60±0.96 μM) as compared to prostate cancer cells PC3 (IC₅₀=10.10±0.59 μM) and immortalized human uroepithelial cell SV-HUC-1 (IC₅₀=18.10±1.03 μM), respectively. NTUB 1 was chose to study the anticancer mechanism of subamolide A in the following experiments. As shown in FIG. 3, subamolide A caused a dose-dependent reduction of growth in NTUB 1 and T24 cells after 72 hours while less cytotoxic effect was observed in PC3 and SV-HUC-1 cells.

V. Subamolide A Induces Apoptosis in NTUB1 Cells

Please refer to FIG. 4( a), after NTUB 1 cells (3×10⁵ cells/well) were treated with 1, 5 and 10 μM of subamolide A for 24 hours, the increased sub-G1 apoptotic fraction was observed in a dose-dependent fashion (5%, 10.8%, and 21.9%, respectively) using flow cytometry when compared with the untreated control.

VI. Quantitative Analysis of Intracellular Reactive Oxygen Species (ROS)

Production of ROS was analyzed by flow cytometry. Briefly, cells were plated in 6-well plates, and dichlorofluorescein diacetate (DCFH-DA, 10 μl) were added to the treated cells 30 minutes prior to harvest. The cells were collected by trypsinization and washed with PBS. The green fluorescence of intracellular DCF (2′,7′-dichlorofluorescein) was then analyzed by FACScan™ flow cytometer with a 525-nm band pass filter. The ROS production efficiency (M1 ratio) was calculated as “[counts of treated sample in M1—counts of control in M1]/counts of control in M1×100”.

ROS causes a wide range of adaptive cellular responses ranging from transient growth arrest to permanent growth arrest, apoptosis, or necrosis, depending on the amount of ROS. Here, the effect of subamolide A on the intracellular ROS level in NTUB1 and SV-HUC-1 cells were evaluated.

Please refer to FIG. 4( b), the ROS M1 ratio of NTUB1 cells was—14.7±0.99%, while that of SV-HUC-1 cells was −11.6±1.41% at 10 μM of subamolide A treatment when compared with the control cells. Subamolide A caused a significant reduction in intracellular ROS level (M1 ratio) but similar changes were observed in both cell lines with increased concentrations. That is, subamolide A reduced the ROS production in NTUB1 and SV-HUC-1 cell lines. The results suggest that alterations in ROS levels do not differentiate cytotoxicity in these cell systems.

VII. Mitochondrial Apoptotic Pathway by Mitochondrial Membrane Potential (MMP; Δψm) measurement

MMP levels were measured by the lipophilic cation JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′,-tetraethylbenzimidazolylcarbocyanine iodide) fluorescent dye. Briefly, cells were plated and treated as the aforementioned conditions. JC-1 (1 μM) was added to the treated cells 30 minutes prior to harvest. The cells were collected by trypsinization and washed with PBS. The red (aggregated JC1; R1 region) and green (monomeric JC1; R2 region) fluorescence signals were analyzed immediately by FACScan™ flow cytometer and Cell Quest™ software.

To determine whether subamolide A induces cytotoxicity by triggering the mitochondrial apoptotic pathway, the changes of mitochondrial membrane potential (Δψm) in subamolide A-treated NTUB 1 cells were measured. It was that subamolide A converted JC-1 from aggregate form (red fluorescence; R1) to monomer form (green fluorescence; R2) indicated the disruption of mitochondrial function at 10 μM of subamolide A treatment (3.52% versus 52.04%).

In addition, please refer to FIGS. 5( a) and 5(b), at a reduced Δψm, subamolide A up-regulated Bax and down-regulated of Bcl-2 expression, resulting in a two-fold increase of Bax/Bcl-2 ratio at 10 μM of subamolide A. Concomitantly, cytochrome c was released from the mitochondria to the cytosol (to 2.3 folds at 10 μM of subamolide A) (FIG. 5( c)). In addition, decreased pro-caspase 3 and the 116 kDa full length PARP (poly (ADP-ribose) polymerase) proteins were observed with the increased concentration of subamolide A, indicating that caspase 3 is activated and cleaved by PARP and demonstrating that subamolide A-induced apoptosis is through activating mitochondrial apoptotic signaling pathways.

VIII. Effect of Subamolide A-Induced Apoptosis in NTUB 1 Cells

Please refer to the immunoblotting spectrum in FIG. 6( a), ERK1/2 (extracellular signal-regulated kinases 1 and 2) but not p38 or JNK (c-Jun N-terminal kinase) was specifically activated by 10 μM of subamolide A at 24 hours in NTUB1 cells. Furthermore, subamolide A induced p53 protein and phosphorylation of p53 at serine 15 (p-p53) in NTUB 1 cells, suggesting that p53 is critical for the induction of apoptosis mediated by subamolide A (FIG. 6( b)).

IV. Combinational Cytotoxicity of Subamolide A with Cisplatin or Gemcitabine on NTUB 1 Cells

Cisplatin (CDDP) and gemcitabine (Gem) are commercialized chemotherapeutic agents, in which the IC₅₀ values of CDDP and Gem to NTUB 1 cells (a seeding number of 1×10³ cells/well at beginning) are 3 μM and 8 nM at 72 hours (data not shown). Please refer to FIG. 7( a), a sub-cytotoxic concentration of CDDP (1 and 3 μM) (or Gem (2 and 8 nM)) was co-treated with various doses of subamolide A (0, 1, 5 and 10 μM) on NTUB1 cells (a seeding number of 1×10³ cells/well at beginning) and cell viability was monitored by the MTT assay and the combination index (CI) was determined for three successive days. It was found that subamolide A showed an effective and significant inhibitory effect on the growth of NTUB 1 cells with both CDDP and Gem (referring to FIGS. 7( a), 7(b), 7(c) and 7(d)), suggesting that subamolide A and CDDP (or subamolide A and Gem) shows synergism on urothelial carcinoma cells (such as NTUB1 cells).

Since subamolide A is the major component of the C. subavenium supercritical CO₂ extract, a more excellent inhibition effect would be obtained by one skilled in the art by co-treating the C. subavenium supercritical CO₂ extract containing subamolide A with CDDP (or Gem) on the urothelial carcinoma cells.

The aforementioned assay was made by combining several concentrations of subamolide A with those of CDDP (or Gem) for analyzing the synergistic effect. The concentrations of subamolide A and CDDP (or Gem) can be arbitrarily adjusted to be administered on NTUB 1 cells or other cell lines or otherwise be administered on the animals with the appropriate physiological conditions (e.g. body weight, age, sex, disease, etc.) and appropriate experimental conditions by one skilled in the art. For instance, in a cellular experiment with NTUB 1 cells at a density of 1×10³ cells/well, the concentration of subamolide A was adjusted as lower than 1 μM, within 1 to 10 μM, or higher than 10 μM, the concentration of CDDP was adjusted as lower than 1 μM, within 1 to 3 μM, or higher than 3 μM, and the concentration of Gem was adjusted as lower than 2 μM, within 2 to 8 μM, or higher than 8 μM. In the animal mode, (1) the appropriate amounts of subamolide A and CDDP (or Gem), (2) the appropriate amounts of the C. subavenium MeOH extract (containing an appropriate amount of subamolide A) and CDDP (or Gem), or (3) the appropriate amounts of the C. subavenium supercritical CO₂ extract (containing an appropriate amount of subamolide A) and CDDP (or Gem) were administered in accordance with animals’ conditions, e.g. body weight, age, sex, disease, etc. Furthermore, the appropriate amounts of subamolide A (in the C. subavenium MeOH extract or the C. subavenium supercritical CO₂ extract) and CDDP (or Gem) administered on the human being could be calculated/reduced by one skilled in the art. The pharmaceutical composition including the appropriate amounts of subamolide A and CDDP (or Gem) could be administered on the animal cells and animals, which include human beings, rodents, other mammals and so on.

In addition, please refer to FIGS. 8( a) and 8(b), it was found that the cytotoxic activity of NTUB 1 cells can be synergistically increased by the combination of the C. subavenium supercritical CO₂ extract (lower than 1 and 2 μg/mL) with CDDP (1 μM) (or Gem of 2 nM), and its synergistic effect was better than subamolide A alone.

While the invention has been described in terms of what is presently considered to be the most practical and preferred Embodiments, it is to be understood that the invention needs not be limited to the disclosed Embodiments. On the contrary, it is intended to cover various modifications and similar arrangements included within the spirit and scope of the appended claims, which are to be accorded with the broadest interpretation so as to encompass all such modifications and similar structures. 

What is claimed is:
 1. A preparation method for a Cinnamomum subavenium extract, comprising steps of: (a) drying a plant of a Cinnamomum subavenium; (b) pulverizing the plant into plural particles; and (c) extracting the plural particles with a supercritical carbon dioxide to obtain the C. subavenium extract comprising a [(3Z,4R,5R)-3-tetradecylidene-4-hydroxy-5-methoxy-5-methylbutanolide] (subamolide A).
 2. The preparation method according to claim 1, wherein the plant has at least one stem, and the step (a) further comprises a step of (a1) drying the at least one stem.
 3. The preparation method according to claim 1, wherein the step (c) is performed at a pressure between 150 bar and 350 bar, a temperature between 45° C. and 55° C., a flow rate of the supercritical carbon dioxide between 4 L/hr and 6 L/hr and a packing density of material between 250 g/L and 320 g/L.
 4. The preparation method according to claim 3, wherein the step (c) is further performed at the pressure of 250 bar, the temperature of 45° C., the flow rate of the supercritical carbon dioxide of 4 L/hr and the packing density of material of 320 g/L.
 5. A pharmaceutical composition of a Cinnamomum subavenium extract for one of a treatment of cancer cells and an growth inhibition of the cancer cells, comprising: a first component having a subamolide A; and a second component being selected from a group consisting of a monoterpene, a sesquiterpene, a sesquiterpene derivative, a saturated fatty acid, a butanolide, a phytosterol, a triterpene, a phytosterone and a combination thereof.
 6. The pharmaceutical composition according to claim 5, wherein the monoterpene is an eugenol.
 7. The pharmaceutical composition according to claim 5, wherein the sesquiterpine is selected from a group consisting of an α-cubebene, an α-bergamotene, a trans-α-bergamotene, a γ-elemene, a β-acoradene, an α-zingiberene, a cis-α-bisabolene, a β-bisabolene, a β-curcumene, a β-amorphene, a trans-α-bisabolene and a combination thereof.
 8. The pharmaceutical composition according to claim 5, wherein the sequiterpene derivative is selected from a group consisting of a cedr-8-ene, an α-curcumene, a nerolidol, a caryophyllene oxide, a humulene-1,2-epoxide, a cubenol, a τ-cadinol, an α-cadinol, an epi-β-cadinol, an epi-β-bisabolol, an epi-α-bisabolol, an α-bisabalol, a 1,10-dihydro nootkatone and a combination thereof.
 9. The pharmaceutical composition according to claim 5, wherein the saturated fatty acid is an ethyl palmitate.
 10. The pharmaceutical composition according to claim 5, wherein the butanolide is selected from a group consisting of an isolinderanolide B, a linderanolide B, a secosubamolide and a combination thereof.
 11. The pharmaceutical composition according to claim 5, wherein the phytosterol is at least one of a β-sitosterol and a γ-sitosterol.
 12. The pharmaceutical composition according to claim 5, wherein the triterpene is a betulin.
 13. The pharmaceutical composition according to claim 5, wherein the phytosterone is a sitostenone.
 14. A pharmaceutical composition, comprising: a subamolide A with a first effective amount; and an ingredient with a second effective amount and being one of an alkylating agent and a nucleoside analog.
 15. The pharmaceutical composition according to claim 14, wherein the subamolide A is a component of a supercritical carbon dioxide extract of a Cinnamomum subavenium.
 16. The pharmaceutical composition according to claim 14, wherein the subamolide A is a component of a methanol extract of a Cinnamomum subavenium.
 17. The pharmaceutical composition according to claim 14, wherein the alkylating agent is selected from a group consisting of a cis-diamminedichloridoplatinum, a cis-diammine(1,1-cyclobutanedicarboxy-lato)platinum (II) and a [(1R,2R)-cyclohexane-1,2-diamine](ethanedioato-O′,O′)platinum (II).
 18. The pharmaceutical composition according to claim 14, wherein the nucleoside analog is selected from a group consisting of a deoxyadenosine analog, a deoxycytidine analog, a deoxyguanosine analog, a deoxythymidine analog, a deoxyuridine analog, a 6-thiohypoxanthine and a fluorouracil.
 19. The pharmaceutical composition according to claim 18, wherein the deoxycytidine analog is a 4-amino-1-(2-deoxy-2,2-difluoro-β-D-erythro-pentofuranosyl)-pyrimidin-2(1H)-on (gemcitabine). 